Methods of pitching yeast for fermentation, and related methods of fermentation and systems

ABSTRACT

The present invention relates to methods and systems of pitching yeast to fermentation reactors. More particularly, the present invention involves pitching yeast from one fermentation tank to at least one additional fermentation tank. Advantageously, yeast can be continuously pitched from fermentor to fermentor for as long as practically desirable.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/741,885, filed Jun. 17, 2015, which claims the benefit of U.S.Provisional Application No. 62/016,481, filed on Jun. 24, 2014, theentire contents of each application which are incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to yeast fermentation to produce analcohol. More particularly, the invention relates to methods and systemsof pitching yeast to fermentation reactors.

BACKGROUND OF INVENTION

Organisms that can convert one or more monosaccharides into one or morebiochemicals such as biofuels are well known. For example, bothgenetically modified (referred to as GM) yeast and non-geneticallymodified (referred to as non-GM) yeast are well known organisms that canconvert sugars into alcohols such as ethanol and butanol viafermentation.

Yeast can be conditioned under conditions similar to those used duringfermentation so as to, e.g., help the yeast be more effective inproducing one or more bioproducts such as ethanol. Oftentimes,conditioning is performed in a propagation system which is typicallymuch smaller in volumetric size as compared to a fermentation system.The conditioned yeast can then be “pitched” (i.e., transferred) from adedicated conditioning and/or propagating tank to a separatefermentation reactor (i.e., a fermentor). Propagation and conditioningtanks can be a source of bacterial contamination which can impact theperformance of yeast during fermentation in a fermentation reactor to anundue degree (e.g., due to bacteria producing undue amounts of lacticacid).

An alternative to pitching yeast from a propagation tank and/orconditioning tank to a fermentation tank includes what is often referredto as “dry-batching.” Dry-batching involves directly pitching yeast intoa single fermentation tank so as to eliminate propagation and/orconditioning tanks. For example, dry-batching can include directlypitching yeast into each batch fermentation tank of a fermentationsystem. Such dry-batching techniques can be very expensive due to theamount of fresh yeast pitched directly into each batch fermentationtank.

There is a continuing need to discover improved methods of pitchingyeast into a fermentation system (e.g., multiple batch fermentationvessels) in a manner that avoids undue bacterial contamination and/orreduces the amount and cost of yeast.

SUMMARY OF INVENTION

The present invention involves pitching yeast from one fermentation tankto at least one additional fermentation tank. Advantageously, yeast canbe continuously pitched from fermentor to fermentor for as long aspractically desirable. Dry-batching into each individual fermentor isnot necessary which can help save on the cost of yeast. If desired,yeast can be provided to the first fermentation tank from a separateyeast propagation and/or a yeast conditioning tank. However, such aseparate yeast propagation and/or yeast conditioning tank is notnecessary. Yeast can be provided to the first fermentation tank viadry-batching. Then, yeast can be pitched from the first fermentationtank to a second fermentation tank; from the second fermentation tank toa third fermentation tank; and so on. Advantageously, because a separateyeast propagation and/or a yeast conditioning tank can be omitted ifdesired, bacterial contamination can be better managed.

According to one aspect of the present invention, a method of pitchingyeast for fermentation includes:

providing a first aqueous composition in a first fermentation reactor,wherein the aqueous composition includes:

-   -   yeast;    -   a slurry including water and a processed plant material        including an amount of at least one monosaccharide;

removing a volume of the first aqueous composition from the firstfermentation reactor; and

providing the removed volume of the first aqueous composition to asecond fermentation reactor.

According to another aspect of the present invention, a fermentationsystem for pitching yeast among two or more fermentation reactorsincludes:

a source of yeast;

a source of a slurry, wherein the slurry includes water and a processedplant material including an amount of at least one monosaccharide;

a first fermentation reactor, wherein the source of yeast and the sourceof the slurry are each in fluid communication with the firstfermentation reactor so that the yeast and slurry can be added to thefirst fermentation reactor to form a first aqueous composition;

a second fermentation reactor in fluid communication with the firstfermentation reactor so that a volume of the first aqueous compositionfrom the first fermentation reactor can be transferred to the secondfermentation reactor to form at least a portion of a second aqueouscomposition, wherein the source of the slurry is in fluid communicationwith the second fermentation reactor so that the slurry can be added tothe second fermentation reactor and combined with the volume of thefirst aqueous composition to form the second aqueous composition.

According to another aspect of the present invention, a fermentationsystem for pitching yeast among two or more fermentation reactorsincludes:

a source of yeast;

a source of a slurry, wherein the slurry includes water and a processedplant material comprising an amount of at least one monosaccharide;

a first reactor, wherein the source of yeast and the source of theslurry are each in fluid communication with the first reactor so thatthe yeast and slurry can be added to the first reactor to form a firstaqueous composition, wherein the first reactor includes a yeastconditioning reactor or an aerobic yeast propagation reactor;

a second reactor in fluid communication with the first reactor so that avolume of the first aqueous composition from the first reactor can betransferred to the second reactor to form at least a portion of a secondaqueous composition, wherein the second reactor includes a fermentationreactor, wherein the source of the slurry is in fluid communication withthe second reactor so that the slurry can be added to the second reactorand combined with the volume of the first aqueous composition to formthe second aqueous composition; and

a third reactor in fluid communication with the second reactor so that avolume of the second aqueous composition from the second reactor can betransferred to the third reactor to form at least a portion of a thirdaqueous composition, wherein the third reactor comprises a fermentationreactor, wherein the source of the slurry is in fluid communication withthe third reactor so that the slurry can be added to the third reactorand combined with the volume of the second aqueous composition to formthe third aqueous composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow diagram of an exemplary fermentation systemaccording to the present invention for pitching yeast from onefermentation reactor to another fermentation reactor.

FIG. 2 shows a flow diagram of another exemplary fermentation systemaccording to the present invention for pitching yeast from onefermentation reactor to another fermentation reactor.

FIG. 3 shows a graph of ethanol concentration at 88 hours for each cycle(i.e., each fermentation reactor) in Example 1.

FIG. 4 shows a graph of residual glucose for each cycle in Example 1.

FIG. 5 shows a graph of residual starch for each cycle in Example 1.

FIG. 6 shows a graph of ethanol production rate for each cycle inExample 1.

FIG. 7 shows a graph of the ethanol production in Example 2.

FIG. 8 shows a graph of the residual sugars (glucose+fructose) inExample 2.

FIG. 9 shows a graph of the lactic acid in the fermentors in Example 2.

FIG. 10 shows a graph of the acetic acid produced in Example 2.

FIG. 11 shows a graph of the ethanol produced in Example 3.

FIG. 12 shows a graph of the residual glucose in Example 3.

FIG. 13 shows a graph of the residual xylose in Example 3.

FIG. 14 shows a graph of acetic acid in the medium after each cycle inExample 3.

DETAILED DESCRIPTION

The present invention relates to yeast fermentation to produce analcohol such as ethanol. More particularly, the invention relates tomethods and systems of pitching yeast to fermentation reactors (i.e.,“fermentors”).

An initial step in fermentation involves “pitching” yeast to afermentation reactor so that the yeast can ferment sugar into alcohol.Pitching yeast means providing an amount of yeast to a fermentationreactor, where the amount of yeast serves as an inoculum in afermentable composition so that the yeast can ferment sugar intoalcohol.

Any of a variety of yeasts, alone or in combination, can be employed asthe yeast in a process as described herein. Such yeasts include wildyeast as well as genetically modified yeast. Suitable yeasts include anyof a variety of commercially available yeasts, such as commercialstrains of Saccharomyces cerevisiae available under the trade names,e.g., Ethanol Red® from LeSaffre or TransFerm® from Mascoma Corporation.Exemplary yeast strains can ferment xylose and/or glucose into analcohol such as ethanol. For example, a useful strain of yeast includesSaccharomyces cerevisiae yeast altered to convert (i.e., ferment) xyloseand glucose to ethanol (i.e., a genetically modified yeast derived froman organism as described in U.S. Pat. No. 7,622,284). As anotherexample, a useful strain of yeast includes Saccharomyces cerevisiaeyeast altered (i.e., genetically modified) to convert (i.e., ferment)xylose, arabinose, and glucose to ethanol.

Useful types of yeast can be introduced (i.e., pitched) into afermentation reactor in any useful form, e.g., as active dry yeast,yeast cream, and combinations thereof.

Yeast can be loaded into a fermentation reactor in any desired amount,e.g., in an amount that can produce a desired amount of an alcohol.Yeast loading can be described in terms of the unit quantity“grams/liter” or “g/L,” which means grams of yeast per liter offermentation medium that the yeast is to be suspended in. A fermentationmedium can include an aqueous composition having at least onemonosaccharide and, optionally one or more additional components usefulin fermentation (e.g., yeast nutrients, bactericides, acids, bases,combinations of these, and the like). For example, a fermentation mediumcan include a slurry such as slurry 115 described below (e.g., corn mashand one or more optional additives (e.g., urea, bactericide, and one ormore enzymes that can convert a polysaccharide to monosaccharides). Insome embodiments, yeast can be pitched to a fermentation reactor inamount of at least 0.01 g/L, at least 0.1 g/L, at least 1.0 g/L, or evenat least 5.0 g/L. In some embodiments, yeast can be pitched to afermentation reactor in an amount in the range from 0.1 to 20 g/L, 0.1to 10 g/L, or even 0.1 to 5 g/L.

The present invention involves pitching yeast from one fermentation tankto at least one additional fermentation tank. Advantageously, yeast canbe continuously pitched from fermentor to fermentor for as long aspractically desirable. For example, yeast can be pitched in such acontinuous serial manner among at least two fermentation tanks, at leastthree fermentation tanks, at least four fermentation tanks, at leastfive fermentation tanks, at least seven fermentation tanks, or even atleast fifteen fermentation tanks. In some embodiments, pitching yeastaccording to the present invention includes pitching yeast among aplurality of fermentation tanks that are physically coupled in series sothat yeast can be pitched serially from, e.g., a first fermentation tankto a second fermentation tank, then from the second fermentation tank toa third fermentation tank, then from the third fermentation tank to afourth fermentation tank, etc.

The yeast that is provided (i.e., pitched) to the first fermentationtank can be “dry-batched” (e.g., described below in connection with FIG.1), provided from a yeast conditioning and/or aerobic yeast propagationtank (e.g., described below in connection with FIG. 2), or a combinationthereof.

In some embodiments, a conventional aerated yeast propagation reactorand a conventional yeast conditioning reactor can be omitted. Instead,yeast can be provided directly (i.e., “dry-batched”) to a firstfermentation tank before pitching an amount of yeast to a secondfermentation tank. As used herein, “dry-batching” means adding a yeastingredient directly to a fermentation tank without first combining theyeast with a conditioning medium and/or propagation medium in a tankseparate from the fermentation tank. An exemplary yeast ingredient fordry-batching includes active dry yeast, yeast cream, and combinationsthereof.

FIG. 1 shows a schematic drawing illustrating an exemplary embodiment ofa method of pitching yeast for fermentation according to the presentinvention. As shown, a source of a slurry 115 is in fluid communicationwith fermentation tank 120 via line 116 so that tank 120 can be filledwith the slurry to a desirable level for fermentation.

Slurry 115 can be any fermentable medium for a yeast. For example,slurry 115 can include an aqueous composition having water and aprocessed plant material. The processed plant material includes anamount of at least one monosaccharide that the yeast can use to convertinto an alcohol. Such monosaccharides are derived from polysaccharidespresent in raw plant material. Further, in addition to including one ormore monosaccharides, a processed plant material can also include one ormore polysaccharides and/or one or more oligosaccharides. Exemplarymonosaccharides include five carbon sugars such as xylose and six carbonsugars such as glucose. Exemplary polysaccharides include cellulose,hemicellulose, and starch that can then be broken down intooligosaccharides and further down to monosaccharides that yeast can useto generate alcohol via fermentation.

Processed plant material can be derived from all or part of any plant.For example, processed plant material can be derived from one or moregrains and/or one or more lignocellulosic substrates and/or starchy rootcrops, tubers, or roots such as sweet potato and cassava. Suitablegrains include maize (corn, e.g., ground whole corn), sorghum (milo),barley, wheat, rye, rice, and millet. Suitable lignocellulosicsubstrates include hardwoods, grasses, softwood, waste paper and pulp,municipal wastes, agricultural wastes such as straws, corn cobs, cornstover, and mixtures thereof. Lignocellulosic feedstock is well knownand includes cellulose, hemicellulose and lignin.

A plant can be processed by any technique so as to provide a processedplant material that includes an amount of at least one monosaccharide.For example, corn grain can be ground (e.g., via dry grinding) into cornflour and then mixed with water and/or backset to make a slurry. Theslurry can be combined with one or more enzymes that can break downstarch in the corn flour into glucose. One preferred technique forprocessing corn grain to be used in fermentation avoids “cooking” and isdescribed in U.S. Pat. No. 7,842,484 (Lewis), wherein said patent isincorporated herein by reference for all purposes.

Another technique for processing a plant to provide a processed plantmaterial that includes an amount of at least one monosaccharide includeshydrolyzing a lignocellulosic substrate to provide a solid componentincluding glucan and lignin and a liquid component including amonosaccharide such as xylose and/or glucose. In some embodiments, thesolid component can be separated from the liquid component so that theliquid component can be used to form at least a portion of the processedplant material for fermentation as described herein. Hydrolyzinglignocellulosic substrates to provide, e.g., xylose and/or glucose iswell-known and is described in, e.g., U.S. Pat. No. 5,424,417 (Torget etal.); U.S. Pat. No. 6,022,419 (Torget et al.); and U.S. Pat. No.8,450,094 (Narendranath et al.), wherein the entireties of said patentdocuments are incorporated herein by reference for all purposes.

Optionally, slurry 115 can include one or more additional components.For example, slurry 115 can include added nutrients (e.g., yeastmicronutrients), urea, bacteriophages, antibiotics, salts, addedenzymes, and the like. Nutrients can be derived from stillage that isadded to slurry 115. Stillage is obtained from distillation of fermentedbeer and is recovered as the bottoms portion of a distillation unit.Stillage can be separated into components including a thin stillagecomponent (also referred to as “backset”) and a wet distillers grainscomponent. Suitable salts can include zinc or magnesium salts, such aszinc sulfate, magnesium sulfate, and the like. Suitable added enzymesinclude those added to conventional processes, such as protease,phytase, cellulase, hemicellulase, exo- and endo-glucanase, xylanase,and the like.

As shown, yeast source 110 is also in fluid communication withfermentation tank 120 so that yeast can be combined with slurry 115 andform a fermentable aqueous composition so that yeast can convert sugarinto an alcohol.

One aspect of the present invention involves removing a volume of theaqueous composition from the first fermentation reactor 120 and pitchingit to a second fermentation reactor 130 via line 125.

The volume of the aqueous composition that is pitched from reactor 120to reactor 130 can serve as an inoculum for fermentation in reactor 130.In some embodiments, the volume of the aqueous composition that ispitched from reactor 120 to reactor 130 includes a volume similar to orthe same as the volume provided by a conventional propagation orconditioning reactor. Exemplary volumes of aqueous compositions that canbe pitched from one fermentation reactor to another fermentation reactorinclude volumes from 5,000 US gallons to less than 100,000 US gallons.In some embodiments, the volume of aqueous compositions that can bepitched from one fermentation reactor to another fermentation reactorincludes a volume from 10,000 US gallons to 40,000 US gallons (e.g.,20,000 US gallons).

The volume of the aqueous composition that is pitched from reactor 120to reactor 130 can be pitched when reactor 120 is full or as reactor 120is filling with slurry 115 and yeast 110. Oftentimes, the volume of thefirst aqueous composition is removed from the first fermentation reactor120 when the first fermentation reactor 120 is substantially filled withthe aqueous composition that includes slurry 115 and yeast 110. In someembodiments, the aqueous composition (slurry 115 and yeast 110) infermentation tank 120 is exposed to conditions in tank 120 for a periodof time to grow and adapt the yeast before removing a volume of theaqueous composition from tank 120 and pitching the aqueous compositionto fermentation tank 130 via line 125.

Adapting (also referred to herein as “conditioning”) yeast meansexposing yeast to conditions that are similar to or the same asconditions used for fermentation so as to help the yeast acclimate whenthe yeast is exposed to a fermentation environment such as in afermentation reactor.

Conditions for conditioning an aqueous composition that includes yeastinclude at least one or more of time, temperature, pH, and stirring. Inexemplary embodiments, yeast can be conditioned in fermentation tank 120at a temperature in the range from 15° C. to 50° C., preferably from 20°C. to 40° C., and even more preferably from 25° C. to 40° C.

In exemplary embodiments, yeast can be conditioned in fermentation tank120 for a period of time of at least 5 hours, at least 8 hours, or evenat least 24 hours. For example, from 5 hours to 100 hours, from 16 hoursto 90 hours, from 5 to 30 hours, or even from 24 to 72 hours.

The aqueous composition in tank 120 including yeast 110 and slurry 115can have a pH that promotes conditioning the yeast. In exemplaryembodiments, the aqueous composition in tank 120 including yeast 110 andslurry 115 can have a pH of about 6 or less, a pH of about 3 to about 6,about 3.5 to about 6, about 4 to about 5, about 4 to about 4.5, or about4.5 to about 5. The initial pH of the aqueous composition can beadjusted by addition of, for example, ammonia, potassium hydroxide,sulfuric acid, phosphoric acid, process waters (e.g., stillage (e.g.,thin stillage (backset)), evaporator condensate (distillate), sidestripper bottoms, and the like), and the like. Further, the pH can beselected so as to promote yeast growth while inhibiting the growth ofcontaminant bacterial strains such as lactic and acetic acid bacteria.For example, a pH in the range of 3.5 to 4.0 may facilitate growth ofyeast while inhibiting the growth and function of contaminatingbacteria.

In some embodiments, it may be desirable to stir the aqueous compositionin fermentation tank 120 during at least the conditioning period so asto keep the components well mixed in the aqueous composition (i.e., theyeast 110 and slurry 115).

Depending on the conditions selected for conditioning (e.g., time,etc.), the aqueous composition in tank 120 may or may not ferment andconvert sugar to alcohol before removing a volume of the aqueouscomposition from the first fermentation reactor 120 and pitching it to asecond fermentation reactor 130 via line 125.

In some embodiments, the aqueous composition in tank 120 may besubjected to conditions so as to produce an amount of alcohol such thatthe volume of the aqueous composition from the first fermentationreactor 120 is not bactericidal. For example, the aqueous composition inthe first fermentation reactor 120 may include an alcohol at aconcentration of less than 15 percent by volume when the volume of theaqueous composition is removed from the first fermentation reactor 120and pitched to second fermentation reactor 130 via line 125.

In some other embodiments, the aqueous composition in tank 120 may besubjected to conditions so as to produce an amount of alcohol such thatthe volume of the aqueous composition from the first fermentationreactor 120 is bactericidal. For example, the aqueous composition in thefirst fermentation reactor 120 may include an alcohol at a concentrationof at least 15 percent (or even at least 19 percent) by volume when thevolume of the aqueous composition is removed from the first fermentationreactor 120 and pitched to second fermentation reactor 130 via line 125.

After yeast is pitched to the second fermentation reactor 130, reactor130 can be filled with slurry 115 via lines 117 and 119 and in a mannersimilar to filling fermentation reactor 120 with slurry 115.

Optionally, as indicated by the dotted lines in FIG. 1, yeast can bepitched from second fermentation reactor 130 to third fermentationreactor 140 in a manner similar to that described above with respect topitching yeast from first fermentation reactor 120 to secondfermentation reactor 130. For example, a volume of the aqueouscomposition from the second fermentation reactor 130 can be removed andpitched to third fermentation reactor 140 via line 135. The volume ofthe aqueous composition that is pitched from reactor 130 to reactor 140can serve as an inoculum for fermentation in reactor 140. After yeast ispitched to the third fermentation reactor 140, reactor 140 can be filledwith slurry 115 via lines 117 and 118 and in a manner similar to fillingfermentation reactor 120 with slurry 115.

Fermentation reactors useful in the present invention are well known andinclude a wide variety of volumetric capacities. Exemplary volumetriccapacities for fermentation reactors such as reactors 120, 130, and 140include from 100,000 US gallons to 750,000 US gallons (e.g., 550,000 USgallons), from 200,000 US gallons to 750,000 US gallons, and even from300,000 US gallons to 750,000 US gallons.

As mentioned above, in some embodiments, a conventional aerated yeastpropagation reactor or a conventional yeast conditioning reactor can beused to pitch yeast to the first fermentation reactor 120 discussedabove instead of or in addition to dry-batching yeast to firstfermentation reactor 120. FIG. 2 shows a schematic drawing illustratingan exemplary embodiment of such a method that uses a conventionalaerated yeast propagation reactor or a conventional yeast conditioningreactor to pitch yeast to a fermentation reactor, where the fermentationreactor is then used to pitch yeast to at least one additionalfermentation reactor. FIG. 2 includes reference characters that are alsoincluded in FIG. 1 to represent substantially the same featuresdiscussed above with respect to FIG. 1. Unless otherwise noted below, adiscussion of said repeated reference characters is not included withrespect to the discussion below of FIG. 2.

For purposes of discussion of the embodiment described in FIG. 2, tank220 represents a conventional vessel for aerobically propagating yeastand/or conditioning yeast (discussed above) for fermentation. Suchvessels are well known and are separate from a fermentation tank so thatthey can be dedicated to the purpose of propagating yeast and/orconditioning yeast for fermentation. Oftentimes, such vessels are afraction of the size of fermentation vessels. For example, such vesselscan have a volumetric capacity in the range from about 1,000 US gallonsto about 75,000 US gallons, or from about 10,000 US gallons to about30,000 US gallons.

As shown in FIG. 2, a source of slurry 115 is in fluid communicationwith tank 220 via line 216 so that tank 220 can be filled with theslurry to a desirable level for propagation and/or conditioning.

Also, as shown in FIG. 2, yeast source 210 is in fluid communicationwith tank 220 so that yeast can be combined with slurry 115 and form anaqueous composition that can be subjected to conditions for yeastpropagation and/or yeast conditioning.

Propagating yeast is generally well known and involves providingconditions to allow yeast to reproduce to provide a larger cell mass ofyeast. Propagating yeast is typically performed by a yeast supplier or ayeast purchaser. In the context of a yeast purchaser, oftentimes thepurchaser will buy an amount of yeast and then propagate the yeastthemselves so as to obtain a larger, more desirable, cell mass of yeastin a more cost effective manner. Exemplary methods of propagating yeastare described in U.S. patent application having Ser. No. 13/798,617 andfiling date Mar. 13, 2013 (Narendranath), wherein the entirety of saidapplication is incorporated herein by reference for all purposes.

In some embodiments, conditions for yeast conditioning are the same asconditions for yeast propagation, although yeast propagation typicallyinvolves aerating the propagation medium to maintain the dissolvedoxygen level at or above the minimum needed for effective yeastpropagation. As described herein, an “aerated yeast propagation” reactorcan provide a level of oxygen to the propagation medium of at least 1volume of air per volume of medium (vvm), preferably at least 0.5 vvm ofair. Yeast conditioning typically does not involve aerating theconditioning medium, especially in the context of using corn mashslurries as the conditioning medium because such slurries can bechallenging to aerate due to their viscosity and presence of particulatematerial.

After conditioning and/or propagation in tank 220, a volume of theaqueous composition from the tank 220 can be removed and pitched tofirst fermentation reactor 120 via line 225. In some embodiments, theentire volume in tank 220 is transferred (i.e., pitched) to reactor 120.The volume of the aqueous composition that is pitched from tank 220 toreactor 120 can serve as an inoculum for fermentation in reactor 120.Exemplary volumes of aqueous compositions that can be pitched from tank220 to reactor 120 include volumes from 5,000 US gallons to less than100,000 US gallons. In some embodiments, the volume of aqueouscompositions that can be pitched from tank 220 to reactor 120 includes avolume from 10,000 US gallons to 40,000 US gallons (e.g., 20,000 USgallons).

After or while the volume of aqueous composition is pitched from tank220 to fermentation reactor 120, reactor 120 can be filled with slurry115 via lines 117 and 116 to a form an aqueous composition that can befermented in reactor 120. According to an aspect of the presentinvention, a volume of the aqueous composition in the first fermentationreactor 120 can be pitched to a second fermentation reactor 130 via line125 in a similar manner as described above with respect to FIG. 1. Thevolume of the aqueous composition that is pitched from reactor 120 toreactor 130 can serve as an inoculum for fermentation in reactor 130.

After yeast is pitched to a fermentation reactor such as reactors 120,130, and 140, fermentation can be performed as desired according to anydesired fermentation protocol. Fermentation is well-known.

After fermentation, alcohol can be recovered from the fermented beervia, e.g., distillation. Distillation is also well-known.

Exemplary methods of fermentation and distillation are described in U.S.Pat. No. 7,842,484 (Lewis), wherein the entirety of said patent isincorporated herein by reference for all purposes.

EXAMPLE 1 Pitching Yeast Continuously from One Fermentor to AnotherFermentor

Example 1 demonstrates that yeast can be pitched continuously from onefermentor to another fermentor in series as similarly described abovewith respect to FIG. 1. Example 1 was conducted in laboratory scale inraw starch (BPXTM) fermentation (i.e., a process that does not “cook”starch prior to fermentation).

Corn flour ground to 95% fines (i.e. >95% of the particles pass througha 0.5 micron sieve) was mixed with backset from a BPX biorefinery toyield a slurry with 35% total solids. The experiment was performed intriplicate. Three 100 mL fermentor bottles were used with 60 mL slurryper bottle. After mixing the corn flour with backset separately in eachfermentor bottle, the slurry in each bottle was pH adjusted to 4.5 usingdilute sulfuric acid or 45% w/w potassium hydroxide. Then, lactoside 247for prevention of bacterial contamination and urea for nitrogen sourceto yeast were added at 2.5 ppm and 4 mM, respectively. A raw starchenzyme blend was added at a dose equivalent to 221 L per 550,000 galfermentor to hydrolyze the starch and form glucose. Then, yeast(Saccharomyces cerevisiae) was inoculated at 0.33 g (dry)/L. The yeastwas a genetically modified yeast that expresses glucoamylase and iscommercially available under the tradename TransFerm® from MascomaCorporation. After inoculation of the yeast, the fermentor bottles wereconditioned in a water bath at 31.1° C. (88° F.). Samples were withdrawnat 24, 48, 72, and 88 h after yeast inoculation and analyzed forethanol, glucose, glycerol, and organic acids using high performanceliquid chromatography (HPLC). The 88 h samples were also analyzed forresidual starch content. At 24 h, a portion (−3.2 g) from each of the 3reactors was transferred to a fresh set of 3 reactors with similar cornmash and additives. This is similar to adding yeast from a yeast prop orconditioning tank in an ethanol producing biorefinery. Then, the 2^(nd)set of 3 reactors was also placed in the water bath at 88° F. (31.1° C.)for 88 h. Sampling and analysis of these reactors were similar to thefirst set. After 24 h of inoculation of the second set of reactors, 3.2g from each reactor were transferred to a third set of reactors. Suchcycles were repeated for about 15 consecutive cycles of continuouslypitching the yeast from one set of reactors to the other every 24 h. Acontrol set of 3 reactors was included. The control set had yeastinoculated to them from 8 h old yeast conditioning tanks. Samples werealso withdrawn from the control set of reactors at 24, 48, 72, and 88 hafter yeast inoculation from the yeast conditioning tanks. Samples wereanalyzed for ethanol, glucose, glycerol, and organic acids. The 88 hsamples were also analyzed for residual starch.

The results showed that such continuous yeast pitching from onefermentor to another fermentor can be successfully carried out for atleast 7 consecutive cycles as evidenced by the ethanol titers achieved,residual glucose and residual starch present after each fermentationcycle (FIGS. 3, 4, and 5). FIG. 3 is a graph showing the ethanolproduced after each cycle. The error bars for each data pointindicate±Standard Deviation from the mean of triplicate fermentations.The thick solid line indicates the average ethanol produced by thecontrol fermentors and the dotted lines indicate the range (±StandardDeviation). FIG. 4 is a graph showing the residual glucose after eachcycle. The error bars for each data point indicate±Standard Deviationfrom the mean of triplicate fermentations. The thick solid lineindicates the average ethanol produced by the control fermentors and thedotted lines indicate the range (±Standard Deviation). FIG. 5 is a graphshowing the residual starch after each cycle. The error bars for eachdata point indicate±Standard Deviation from the mean of triplicatefermentations. The thick solid line indicates the average ethanolproduced by the control fermentors and the dotted lines indicate therange (±Standard Deviation). Additionally, no major differences wereobserved in the rates of ethanol production rate during each cycle (FIG.6) which is indicative of no major changes in yeast populations from onecycle to another. The error bars for each data point indicate±StandardDeviation from the mean of triplicate fermentations. The black dottedline indicates the rate of ethanol production in the control fermentors.Moreover, the rate of ethanol production observed in each fermentationcycle was similar to that of the control. Such a process of continuouslypitching yeast from one fermentor to another fermentor may significantlyreduce the yeast cost for any ethanol biorefinery.

EXAMPLE 2

Example 2 was conducted at pilot scale facility in 20,000 galfermentors. In the pilot plant, corn flour was mixed with backset andother process waters to make up a slurry at 32-34% fill solids. A rawstarch enzyme blend, BPX10.5C, was added to the slurry at the requireddose that would provide 0.32 AGU (alpha glucosidase unit)s/g dry solidsand 0.03 FAU-F (fungal alpha amylase unit)s/g dry solids. The slurry(about 800 gal), was pH adjusted to 4.3 and was fed to a yeastprop/conditioning tank to which lactoside 247™ was added at 2-5 ppm andurea added at ˜300 ppm. The same slurry also was used to fill thefermentor tanks. Ethanol Red® active dry yeast was used during thistrial. The fermentor initially was pitched with yeast grown/conditionedin the yeast conditioning tank for 10-12 hours. After 16 h, about 5% ofthe volume of the fermentor was transferred to the next fermentor. This5% serves as the conditioned yeast inoculum in the second fermentor. Theprocess was then continued from the second to the third fermentor and soon so that a series of batch fermentors can effectively operatecontinuously because yeast was pitched after successive 16 hour timeblocks from the most recently “pitched to” fermentor to a new fermentorto start a new fermentation process over a total time period where abatch fermentation process is always in process. While the batchfermentors were continuously pitched with content from the previousfermentor at 16 hours of conditioning, a few variations were tested suchas increasing the pH of the slurry, reducing the transfer volume from 5%to 2.5%, and increasing the fill solids. This operation was continuedsuccessfully in the pilot plant for over 50 fermentors with yeast fromone single initial yeast conditioning tank. Such an operation caneliminate any variations caused due to conditioning yeast for each batchand also quite significantly reduce the fresh yeast usage. It is alsolikely that the yeast strain eventually may be adapted to the specificprocess. The results of ethanol produced after 78 h of fermentation,residual glucose, lactic and acetic acids from this trial are shown inFIGS. 7 through 10. FIG. 7 is a graph showing the ethanol produced inthe fermentors pitched with yeast continually from one to the otherstarting originally from one yeast conditioning tank in batch 1. FIG. 8is a graph showing the residual sugars (glucose+fructose) after 78 h inthe fermentors pitched with yeast continually from one to the otherstarting originally from one yeast conditioning tank in batch 1. FIG. 9is a graph showing lactic acid in the fermentors pitched with yeastcontinually from one to the other starting originally from one yeastconditioning tank in batch 1. FIG. 10 is a graph showing acetic acidproduced in the fermentors pitched with yeast continually from one tothe other starting originally from one yeast conditioning tank in batch1.

The results in Example 2 from the pilot study confirms both thescalability and the resiliency of the continuous yeast pitching to abatch fermentation process for ethanol production in a dry grindfacility.

EXAMPLE 3

Example 3 demonstrates that genetically modified yeast capable ofmetabolizing xylose can successfully be pitched continuously from onefermentor to another in series. Dilute acid pretreated corn stover,pretreated at a pilot facility using 0.59% acid was used for this. BothC6 solids and C5 liquor from acid pretreatment were mixed to form aslurry of 17% solids, pH adjusted to 5.5, and saccharified at 50° C.using the appropriate cellulase mix (CTec3) for 120 h. Aftersaccharification, the broth was stored at 4° C. Post saccharification,the glucose and xylose in the medium were measured at 4.81 and 4.13%w/v, respectively. The experiment was performed in duplicate. Two, 125mL Erlenmeyer flasks were used with 50 mL of the saccharified broth perflask. After dispensing 50 mL of the broth to each flask, the pH of thematerial in each flask was adjusted to 5.5 using ammonium hydroxide.Then, lactoside 247™ was added at 5 ppm to each flask to prevent anybacterial contamination. Then, the genetically modified yeast grown onYeast Extract, Peptone media supplemented with glucose (10 g/L) andxylose (20 g/L) was harvested, washed, and was inoculated to the flasksat 1 g (dry yeast)/L per flask. After inoculation of the yeast, theflasks were placed in a water bath shaker set at 32° C. (89.6° F.), and150 rpm. Samples were withdrawn at 24, and 48 h after yeast inoculationand analyzed for ethanol, glucose, xylose, and acetic acid using highperformance liquid chromatography. At 24 h, a portion (−5.0 g) from eachof the 2 flasks was transferred to a fresh set of 2 flasks with 50 mL ofthe same saccharified broth adjusted to pH 5.5 using ammonium hydroxide.This would be similar to adding the yeast from a yeast propagation tankat a cellulosic ethanol production facility. Then, the second set offlasks were placed in the water bath at similar conditions. Such cycleswere repeated for about 10 consecutive cycles of continuously pitchingyeast from one set of flasks to the other every 24 h. The results ofethanol produced after 48 h, residual sugars and acetic acid from thestudy are shown in FIGS. 11 through 14. FIG. 11 is a graph showingethanol produced after each cycle in a continuous yeast pitch type offermentation in a lignocellulosic biomass to ethanol process. The errorbars indicate±Standard Deviation from the mean of duplicatefermentations. FIG. 12 is a graph showing residual glucose after eachcycle in a continuous yeast pitch type of fermentation in alignocellulosic biomass to ethanol process. The error barsindicate±Standard Deviation from the mean of duplicate fermentations.FIG. 13 is a graph showing residual xylose after each cycle in acontinuous yeast pitch type of fermentation in a lignocellulosic biomassto ethanol process. The error bars indicate±Standard Deviation from themean of duplicate fermentations. FIG. 14 is a graph showing acetic acidin the medium after each cycle in a continuous yeast pitch type offermentation in a lignocellulosic biomass to ethanol process. The errorbars indicate±Standard Deviation from the mean of duplicatefermentations.

The results in Example 3 showed that the continuous yeast pitching fromone fermentor to another fermentor can be successfully carried out forat least up to 10 cycles even in the lignocellulosic biomass to ethanolprocess. In the cycles where the residual xylose appeared to go up over0.5% w/v, this can be reduced by increasing fermentation time by anadditional 12-24 h.

What is claimed is:
 1. A method of fermenting alcohol, the methodcomprising: providing a first aqueous composition in a firstfermentation reactor, wherein the first aqueous composition comprisesyeast, water, processed plant material comprising milled grain orlignocellulosic biomass, and one or more enzymes that can break down theprocessed plant material to yield one or more monosaccharides;fermenting the first aqueous composition in the first fermentationreactor to form a first beer composition comprising alcohol; providing asecond aqueous composition in a second fermentation reactor, wherein thesecond aqueous composition comprises, water, processed plant materialcomprising milled grain or lignocellulosic biomass, and one or moreenzymes that can break down the processed plant material to yield one ormore monosaccharides, and a fraction of the first beer compositionincluding yeast and alcohol from the first beer composition; fermentingthe second aqueous composition in the second fermentation reactor toform a second beer composition comprising alcohol; recovering alcoholfrom the first beer composition from the first fermentation reactor; andrecovering alcohol from the second beer composition from the secondfermentation reactor.
 2. The method of claim 1, wherein adding the yeastto the first fermentation reactor comprises dry-batching yeast to thefirst fermentation reactor.
 3. The method of claim 1, wherein, prior toremoving the fraction of the first beer composition from the firstfermentation reactor, at least the fraction of the first aqueouscomposition is exposed to a temperature in the range from 15° C. to 50°C. for a time period in the range from 5 to 30 hours.
 4. The method ofclaim 1, wherein the volume of the first beer composition is removedfrom the first fermentation reactor at least 8 hours after the firstaqueous composition is provided in the first fermentation reactor. 5.The method of claim 1, wherein the fraction of the first beercomposition is removed from the first fermentation reactor after thefirst fermentation reactor is filled with the first aqueous composition.6. The method of claim 1, wherein the yeast converts at least xylose andglucose to alcohol.
 7. The method of claim 1, further comprising,fermenting the second aqueous composition in the second fermentationreactor to form a second beer composition comprising alcohol; removing afraction of the second beer composition from the second fermentationreactor to a third fermentation reactor; and continuing to ferment thesecond aqueous composition in the second fermentation reactor after theremoving the second beer composition.
 8. The method of claim 1, furthercomprising, prior to providing the first beer composition in the firstfermentation reactor: providing an initial pitching aqueous compositionin a yeast conditioning reactor and/or an aerobic yeast propagationreactor, wherein the initial pitching aqueous composition comprises:yeast; a slurry comprising water and a processed plant materialcomprising an amount of at least one monosaccharide; and removing theinitial pitching aqueous composition from the yeast conditioning reactorand/or aerobic yeast propagation reactor to form the first aqueouscomposition that is provided to the first fermentation reactor.
 9. Themethod of claim 8, wherein the processed plant material comprises milledgrain or lignocellulosic biomass.
 10. The method of claim 8, wherein,prior to removing the initial pitching aqueous composition from theyeast conditioning reactor and/or aerobic yeast propagation reactor, theinitial pitching aqueous composition in the yeast conditioning reactorand/or aerobic yeast propagation reactor is exposed to a temperature inthe range from 15° C. to 50° C. for a time period in the range from 5 to30 hours.
 11. The method of claim 8, wherein the yeast conditioningreactor and/or the aerobic yeast propagation reactor has a capacity ofless than 100,000 gallons and the first and second fermentation reactorseach have a capacity greater than 100,000 gallons.
 12. The method ofclaim 1, wherein the fraction of the first beer composition from thefirst fermentation reactor has a volume in the range from 10,000 gallonsto 40,000 gallons and the first aqueous composition that is fermented inthe first fermentation reactor has a volume in the range from 100,000gallons to 750,000 gallons.
 13. The method of claim 1, wherein thefraction of the first beer composition is from 1.3% by volume to 13.3%by volume based on the total volume of the first aqueous composition.14. The method of claim 1, wherein fermenting the first aqueouscomposition in the first fermentation reactor occurs for a total timeperiod in the range from 25 to 150 hours.
 15. The method of claim 1,wherein wherein the fraction of the first beer composition is removedfrom the first fermentation reactor from 8 hours to 24 hours after thefirst aqueous composition is provided in the first fermentation reactor;wherein the first beer composition comprises an alcohol at aconcentration of less than 15 percent by volume when the fraction of thefirst beer composition is removed from the first fermentation reactor.16. A fermentation system for pitching yeast among two or morefermentation reactors, the system comprising: a source of yeast; asource of a slurry, wherein the slurry comprises water and a processedplant material comprising an amount of at least one monosaccharide; afirst fermentation reactor, wherein the source of yeast and the sourceof the slurry are each in fluid communication with the firstfermentation reactor so that the yeast and slurry can be added to thefirst fermentation reactor to form a first aqueous composition; a secondfermentation reactor in fluid communication with the first fermentationreactor so that a volume of the first aqueous composition from the firstfermentation reactor can be transferred to the second fermentationreactor to form at least a portion of a second aqueous composition,wherein the source of the slurry is in fluid communication with thesecond fermentation reactor so that the slurry can be added to thesecond fermentation reactor and combined with the volume of the firstaqueous composition to form the second aqueous composition.
 17. Thefermentation system of claim 16, further comprising a third fermentationreactor in fluid communication with the second fermentation reactor sothat a volume of the second aqueous composition from the secondfermentation reactor can be transferred to the third fermentationreactor to form at least a portion of a third aqueous composition,wherein the source of the slurry is in fluid communication with thethird fermentation reactor so that the slurry can be added to thirdfermentation reactor and combined with the volume of the second aqueouscomposition to form the third aqueous composition.
 18. A method offermenting alcohol, the method comprising: providing a first aqueouscomposition in a first fermentation reactor, wherein the first aqueouscomposition comprises yeast, processed plant material comprising milledgrain or lignocellulosic biomass, and one or more enzymes that can breakdown the processed plant material to yield one or more monosaccharides;fermenting the first aqueous composition in the first fermentationreactor to produce a first alcohol concentration; providing a secondaqueous composition in a second fermentation reactor, wherein the secondaqueous composition comprises processed plant material comprising milledgrain or lignocellulosic biomass, and one or more enzymes that can breakdown the processed plant material to yield one or more monosaccharides,and a fraction of the first aqueous composition comprising the firstalcohol concentration and including yeast and alcohol removed from thefirst fermentation reactor; further fermenting the first aqueouscomposition in the first fermentation reactor to produce a secondalcohol concentration greater than the first alcohol concentration;fermenting the second aqueous composition in the second fermentationreactor to produce alcohol; recovering alcohol from the first aqueouscomposition comprising the second alcohol concentration and recoveringalcohol from the second aqueous composition.
 19. The method of claim 18,wherein the first alcohol concentration is less than 15 percent byvolume of the first beer composition.
 20. The method of claim 18,wherein the fraction of the first beer composition is from 1.3% byvolume to 13.3% by volume based on the total volume of the first aqueouscomposition.